This invention relates to an apparatus and method for capillary transfer of macromolecules to a membrane.
The fractionation and analysis of biochemical macromolecules including DNA, RNA and protein molecules is often assisted by a technique known as slab gel electrophoresis. This technique involves the use of an electric field to induce the migration and separation of different molecular species in aqueous buffered gels. Such electrophoretic gels are most often formed using electrically neutral polymers including agarose and polyacrylamide. Agarose gels are cast from reversible thermosetting sols whereas polyacrylamide gels are typically polymerized and cross-linked in place and are not thermally reversible. Following electrophoretic fractionation of macromolecules in a slab gel, it is often useful, for purposes of analysis, to transfer the macromolecules from the flat gel (approximately 0.1-1.0 cm thick) onto a thin juxtaposed membrane (typically consisting of microporous nitrocellulose or nylon material). The process of transferring macromolecules such as DNA from a slab gel to a microporous membrane usually involves a capillary blotting procedure in which a salt-containing buffer (e.g., 1.5M NaCl, 0.15M Tris buffer) is fed to the underside of the slab gel. Capillary transport of the buffer solution carries the macromolecules from the gel, upward to the overlayed microporous receiving membrane where the macromolecules are captured. A carefully positioned and weighted stack of absorbent paper is placed on top of the membrane to provide liquid wicking action allowing a substantial volume of buffer to move uniformly upward through the blotting sandwich containing the gel, membrane, and stack of absorbent paper. Air bubbles in the structure must be avoided since any which are accidentally trapped in the blotting sandwich, above or below the slab gel, tend to locally block capillary transport of buffer, compromising the fidelity and quality of the resulting molecular blot. Maniatis et al. in Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, N.Y.) provide a detailed description and protocol describing the molecular blotting procedure. A diagram from this prior art reference is provided in the Figure as illustrative of the method. When this blotting procedure is applied to the transfer of DNA molecules from a gel, it is known as Southern blotting. When the procedure is used for RNA and protein molecules it is known as northern and western blotting respectively.
Absorbent materials utilized in capillary blotting pads for transfer of macromolecules to microporous membranes include paper towels, filter paper sheets and paper pads.